T cell receptor gene transfer to target the Epstein-Barr virus-associated malignancy nasopharyngeal carcinoma

Epstein-Barr Virus (EBV)-positive post-transplant lymphoproliferative disease (PTLD) can be successfully treated by infusing T cells reactivated in vitro with EBV-transformed B lymphoblastoid cell lines (LCLs). T cell lines generated in this way are dominated by reactivities to the EBNA 3 viral proteins. However, such an approach may not be appropriate to treat other more common EBV-associated malignancies since, unlike PTLD, these tumours do not express the immunodominant EBNA 3 proteins; instead viral protein expression is restricted to relatively weak antigens. Furthermore, generating such T cell lines can take many weeks of in vitro culture. T cell receptor (TCR) gene transfer offers an alternative approach whereby T cells of defined specificity can be generated rapidly and reliably from all patients. We are exploring this method to treat undifferentiated Nasopharyngeal carcinoma (NPC), a tumour that occurs at high frequency in Southeast Asia (incidence >20/100,000 population) and where >50% of cases express the EBV protein LMP2. To ensure TCR gene transfer could be widely applicable to treat this cancer we have focussed on the T cell response to an LMP2 epitope that is restricted through HLA A*1101, an allele carried by >50% of the Chinese population. Furthermore the target epitope sequence is conserved in EBV strains present in NPC tumours. Having generated a high avidity T cell clone specific for this epitope the genes encoding the TCR a and b chain were cloned into the same MP71 retroviral vector, separated by a self-cleaving 2A peptide to ensure equal expression. Retrovirus was generated using Phoenix A packaging cells and following stimulation with anti-CD3 antibody, T cells from five A*1101+ donors (including an NPC patient) were transduced with this virus. Three days later, HLA:peptide pentamer staining detected the transferred TCR in 12-17% of CD8+ T cells. Staining for the b chain indicated that mispairing with endogenous a chains can occur in 0-8% of T cells. The TCR-transduced T cells expanded rapidly in vitro in response to antigen, showed high avidity for the target peptide (10-10M), and lysed autologous fibroblasts expressing LMP2. Significantly, they also recognised EBV-transformed LCLs which express physiological levels of LMP2 in an HLA A*1101-restricted manner. Preliminary data indicate that some CD4+ T cells transduced with this TCR also recognised A11.01+ LCLs suggesting they might provide a helper function in vivo to aid persistence of LMP2-specific CD8+ effectors.